CN1987427A - Method and system for detecting antibiotics in milk product based on surface plasma resonant - Google Patents
Method and system for detecting antibiotics in milk product based on surface plasma resonant Download PDFInfo
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- CN1987427A CN1987427A CN 200610130646 CN200610130646A CN1987427A CN 1987427 A CN1987427 A CN 1987427A CN 200610130646 CN200610130646 CN 200610130646 CN 200610130646 A CN200610130646 A CN 200610130646A CN 1987427 A CN1987427 A CN 1987427A
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Abstract
The disclosed detection method includes steps: using SPR optical method to measure refractive indexes of milk products with known different contents of antibiotic; making correspondence between magnitude of measured index and contents of antibiotic in milk products; comparing the measured index of milk product without knowing antibiotic concentration with the indexes of milk products with knowing contents of antibiotic so as to implement detecting contents of antibiotic in milk products. The detection system includes control sect connected to the data process part, display part, injection unit, reversal valve, and SPR sensor. The injection unit, the reversal valve, the SPR sensor, and waste liquor pool are connected in sequence. Features are: simplified detecting steps, short measuring time, high measuring resolution and precision etc.
Description
Technical field
The present invention relates to a kind of food medicament residue detection technique.Particularly relate to a kind of to antibiotic content in the dairy produce can carry out easily and fast and accurately detect based on detection of antibiotics method and system in the surface plasma body resonant vibration dairy products.
Background technology
Microbiotic is the medicine of a kind of sterilization, sterilization and anti-inflammatory, is widely used in the treatment of numerous disease.If antibiotic residue is in milk, long-term eating will cause human body to antibiotic immunity, and be unfavorable to health, may cause that also the people to microbiotic allergy produces allergic reaction.Simultaneously, also the anti-medicine bacterium of cultivation that can be indirect increases the treatment of diseases difficulty.
Antibiotic-free is residual to be one of most important index of dairy food quality safety, also is index the harshest in the international dairy products trade, in World Health Organization's regulation raw material milk and the sterilized milk antibiotic residue must not be arranged.America and Europes etc. have just forbidden the milk listing that antibiotic residue exceeds standard many years ago, and the fresh cow's milk industry standard of pollution-free food has been implemented in the issue of China Ministry of Agriculture, for the sanitary index of fresh milk, clearly stipulates the terrific microbiotic that detects.Aspect the antibiotic food safety detection, all will consume a large amount of funds every year in milk, and the milk industry company that data presentation is big all will buy Snap (euzymelinked immunosorbent assay (ELISA)) kit of 100-500 ten thousand every year, in order to detect Determination of antibiotic in milk content.The detection method of antibiotic residue roughly is divided three classes in the fresh milk at present, is respectively: bioassay method (microbiological assay and radioreceptor assay etc.), physico-chemical analysis method (spectral method and chromatogram and coupling technique etc.), immunization (radioimmunology, fluorescent immune method and euzymelinked immunosorbent assay (ELISA) etc.).TTC method (national standard method) is the test stone (GB4689.27-94) of antibiotic residual quantity in the fresh milk formulated of China, belong to bioassay method, its measuring principle is based on the inhibiting effect of microbiotic to microorganism, it has expense advantage low and that easily realize, shortcoming is length consuming time, requires operating personnel to have the preparation of bacterium liquid in certain professional knowledge and the experimentation and all strict working specification of observing of control of water-bath process, otherwise be prone to false positive, so that the instability of assay occurs; Highly effective liquid phase chromatography detection method, it is a kind of physics and chemistry detection method, be to utilize the special reaction that group had in the antibiotic molecule to measure its content, this method can detection of antibiotics concrete content, susceptibility is higher, but the trace routine complexity, expense is higher, need to buy checkout equipments such as chromatograph, be not suitable for small-sized control laboratory and use.Snap method (euzymelinked immunosorbent assay (ELISA)), belong to immunization, it is a kind of method that the susceptibility and the immunoreactive specificity of antigen-antibody of zymochemistry reaction are combined, its susceptibility and specificity are good, that detects is highly sensitive, has semiquantitative reading, can monitor the situation of pasture medication, detecting instrument has good stability, as a result the reappearance height, whole testing process is simple and convenient, but this technology must rely on external import, needs the disposal reagent kit of acquisition price costliness, and cost is higher, be unfavorable for widespread usage, can't realize the daily control of antibiotics in milk products.
In a word, the traditional detection method of antibiotic content is many in the milk, but the operation that has is loaded down with trivial details, the requirement for experiment condition that has is very high, the proving time that has is oversize, the consumptive material expense that has is too high, and these weak points have caused economical and temporal loss not only for dairy produce manufacturing enterprise, and testing result usually also can be subjected to the influence of factors such as experiment material and manual operation.
Summary of the invention
Technical matters to be solved by this invention is, provide a kind of to antibiotic content in the dairy produce can carry out easily and fast and accurately detect based on detection of antibiotics method and system in the surface plasma body resonant vibration dairy products.
The technical solution adopted in the present invention is: a kind of based on detection of antibiotics method and system in the surface plasma body resonant vibration dairy products.Wherein, based on detection of antibiotics method in the surface plasma body resonant vibration dairy products, be to utilize the SPR optical means, measure the refractive index of the antibiotic dairy produce of known different content, and it is the size of the refractive index that measures is corresponding with antibiotics in milk products content, the refractive index of the refractive index of the dairy produce sample of the unknown antibiotic concentration that will measure and the antibiotic dairy produce of known different content compares again, realizes the detection of antibiotic content in the dairy produce.Specifically be a kind of method that adopts in the indirect measurement method of the direct measuring method of surface plasma body resonant vibration refractive index and surface plasma body resonant vibration refractive index, realize detection antibiotic content in the dairy produce.
The direct measuring method of described surface plasma body resonant vibration refractive index is directly to feed testing sample on the golden film surface of SPR, makes sample directly contact golden film surface and measures, thereby realize antibiotics in milk products qualitative detection and analysis.Include following steps:
(1) feeds deionized water, obtain the stable measuring basis of refractive index; (2) the dairy produce sample of the known different antibiotic contents of preparation by surface plasmon resonance measurement amount refractive index size, obtains the pairing refractive index value of different antibiotic concentrations; (3) set up the typical curve of antibiotic content in the dairy produce sample, and application of mathematical method sets up the theoretical model of antibiotic content, form forecast model; (4) refractive index of the dairy produce of the unknown antibiotic content of measurement, measured result is brought into the forecast model of in the 3rd step, setting up, calculate antibiotic content wherein, and also at the otherness between the different dairy produces, and when test environment temperature and the otherness of forecast model temperature revise, thereby predicted the outcome.
The indirect measurement method of described surface plasma body resonant vibration refractive index, after being antibody by microbiotic selective adsorption to be measured in golden film surface is bound dairy produce, improve specificity, measure the refractive index of dairy produce again and obtain the microbiotic of dairy produce, realize that the accurate quantification of antibiotic content in the dairy produce detects.Include following steps:
(1) utilize BSA absorption, amine to connect the golden film surface that antibody is attached to SPR; (2) feed deionized water, obtain the stable measuring basis of refractive index; (3) the dairy produce sample of the known different antibiotic contents of preparation by surface plasmon resonance measurement amount refractive index size, obtains the pairing refractive index value of different antibiotic concentrations; (4) set up the typical curve of antibiotic content in the dairy produce sample, and application of mathematical method sets up the theoretical model of antibiotic content, form forecast model; (5) refractive index of the dairy produce of the unknown antibiotic content of measurement, measured result is brought into the forecast model of in the 4th step, setting up, calculate antibiotic content wherein, and also at the otherness between the different dairy produces, and when test environment temperature and the otherness of forecast model temperature revise, thereby predicted the outcome.
Wherein, the described BSA of utilization adsorbs, amine connects the golden film surface that antibody is attached to SPR, is to be finished by following steps:
(1) utilizes the BSA physical adsorption characteristic, evenly be applied to golden film surface, add 4: 1 EDC, NHS, and utilize fourth disuccinic acid acid anhydride that BSA carry out carboxylic acidization, form the carboxyl albumin layer, become the connection tie of golden film and antibody protein;
(2) utilize EDC, NHS that the carboxyl albumin layer on golden film surface is handled, form NHS ester activated protein layer, make antibody capable and BSA carry out amine and connect, thereby be attached to uniformly on the golden film.
When the variations in refractive index of the dairy produce sample of measuring unknown antibiotic concentration, all adopt the hyperchannel difference measurement, promptly in a passage, feed deionized water as measuring basis, feed sample to be measured in another passage simultaneously, two groups of data that record are carried out difference processing, thereby the result is revised.
All ingredients in the time of will experimentizing in measurement, sample kept 15-30 minute in 20-25 ℃ water, to guarantee that identical temperature is arranged, reagent, sample feed among the SPR with speed 5-10 μ l/min speed during measurement, continue 5-10 minute, obtain best test curve.
Based on detection of antibiotics system in the surface plasma body resonant vibration dairy products, include: be used for detecting golden film surface liquid change of refractive, gather the spr sensor part of corresponding data; Be used for analyzing the data that the spr sensor part is gathered, the data analysis of gathering is handled, draw the data processing section of the size of corresponding curve and antibiotic concentration; Be used for showing intuitively the display device part of measurement result and corresponding measuring condition; With constant rate of speed reagent is injected the injection device part of spr sensor part on request by reversal valve; Undertaken by the order of setting in order to control above-mentioned various piece, realize the control section of the automatic measurement of sample; Collect the waste liquid pool of waste liquid; Wherein, control section partly links to each other with data processing section, display device part, injection device part, reversal valve, spr sensor respectively, and injection device part, reversal valve, spr sensor part, waste liquid pool link to each other successively.
Also be provided with by the golden film surface of reversal valve in the spr sensor part and inject a kind of of strong acid and highly basic, golden film surface is cleaned, golden film surface capable of repeatedly is used realize the repeatedly cleaning device of precision measurement sample, the liquid feeding end of described cleaning device links to each other with control section, and outlet end links to each other with reversal valve.
Described spr sensor partly is formed with the fluid passage more than 1 or 1, measures simultaneously in order to realize several samples.
Of the present invention based on detection of antibiotics method and system in the surface plasma body resonant vibration dairy products, it is loaded down with trivial details to have overcome existing detection method operation, the requirement for experiment condition height, proving time is oversize, shortcoming and defect such as device and reagent costliness, binding by antibody protein and golden film, can take multiple measurements repeatedly, need not mark, simplified testing procedure, reduced test agent, it is short to have the required time of measurement, Measurement Resolution and measuring accuracy height, measurement result reliability height, advantages such as required consumptive material is few, have repeatedly used feature, reduced the detection cost.Can realize accurate, the online detection of antibiotic content in the dairy produce.
Description of drawings
Fig. 1 is the detection system that realizes the method for the invention;
Fig. 2 is the structural representation of surface plasma resonance sensor gold film surface adsorption antibiotic molecule of the present invention.
Wherein:
1: data processing section 2: control section
3: display device part 4: the injection device part
5: reversal valve 6:SPR Sensor section
7: waste liquid pool 8: fluid passage
9: golden film surface 10:BSA (bovine serum albumin)
11: amine connects 12: the antibody protein molecular layer
13: antibiotic molecule
Embodiment
Make a detailed description based on detection of antibiotics method and system in the surface plasma body resonant vibration dairy products of the present invention below in conjunction with embodiment.
Of the present invention based on detection of antibiotics method in the surface plasma body resonant vibration dairy products, be to utilize SPR (SurfacePlasmon Resonance:SPR surface plasma body resonant vibration) optical means, measure the refractive index of the antibiotic dairy produce of known different content, and it is the size of the refractive index that measures is corresponding with antibiotics in milk products content, the refractive index of the refractive index of the dairy produce sample of the unknown antibiotic concentration that will measure and the antibiotic dairy produce of known different content compares again, realizes the detection of antibiotic content in the dairy produce.
Detection method is specifically: adopt the direct measuring method of surface plasma body resonant vibration refractive index, or the indirect measurement method of surface plasma body resonant vibration refractive index, realize detection to antibiotic content in the dairy produce.
The direct measuring method of described surface plasma body resonant vibration refractive index is directly to feed testing sample on the golden film surface of SPR, makes sample directly contact golden film surface and measures, thereby realize antibiotics in milk products qualitative detection and analysis.Include following steps:
(1) feeds deionized water, obtain the stable measuring basis of refractive index;
(2) the dairy produce sample of the known different antibiotic contents of preparation by surface plasmon resonance measurement amount refractive index size, obtains the pairing refractive index value of different antibiotic concentrations;
(3) set up the typical curve of antibiotic content in the dairy produce sample, and application of mathematical method sets up the theoretical model of antibiotic content, form forecast model;
(4) refractive index of the dairy produce of the unknown antibiotic content of measurement, measured result is brought into the forecast model of in the 3rd step, setting up, calculate antibiotic content wherein, and also at the otherness between the different dairy produces, and when test environment temperature and the otherness of forecast model temperature revise, thereby predicted the outcome.
The indirect measurement method of described surface plasma body resonant vibration refractive index, after being antibody by microbiotic selective adsorption to be measured in golden film surface is bound dairy produce, improve specificity, measure the refractive index of dairy produce again and obtain the microbiotic of dairy produce, realize that the accurate quantification of antibiotic content in the dairy produce detects.Include following steps:
(1) utilize BSA absorption, amine to connect the golden film surface that antibody is attached to SPR;
(2) feed deionized water, obtain the stable measuring basis of refractive index;
(3) the dairy produce sample of the known different antibiotic contents of preparation by surface plasmon resonance measurement amount refractive index size, obtains the pairing refractive index value of different antibiotic concentrations;
(4) set up the typical curve of antibiotic content in the dairy produce sample, and application of mathematical method sets up the theoretical model of antibiotic content, form forecast model;
(5) refractive index of the dairy produce of the unknown antibiotic content of measurement, measured result is brought into the forecast model of in the 4th step, setting up, calculate antibiotic content wherein, and also at the otherness between the different dairy produces, and when test environment temperature and the otherness of forecast model temperature revise, thereby predicted the outcome.
In two kinds of above-mentioned detection methods, when the variations in refractive index of the dairy produce sample of measuring unknown antibiotic concentration, all adopt the hyperchannel difference measurement, promptly in a passage, feed deionized water as measuring basis, feed sample to be measured in another passage simultaneously, two groups of data that record are carried out difference processing, thereby the result is revised.
In testing process, all ingredients, sample when experimentizing kept 15-30 minute in 20-25 ℃ water, and to guarantee that identical temperature is arranged, reagent, sample feed among the SPR with speed 5-10 μ l/min speed during measurement, continue 5-10 minute, obtain best test curve.
Be example with the content that detects microbiotic-ampicillin in the milk below, set forth the detailed detection step of direct measuring method of surface plasma body resonant vibration refractive index:
(1) initialization system in air obtains spr sensor background light intensity, makes the sensor optimization;
(2) in surface plasma resonance sensor gold film surface, feed deionized water by injection device, obtain the stable measuring basis of refractive index (1.333);
(3) preparation ampicillin content is 2500.0 μ gl
-1Milk sample, inject this sample by injection device to surface plasma resonance sensor gold film surface, obtain corresponding refractive index curve;
(4) feed deionized water to surface plasma resonance sensor gold film surface, remove the residual liquid on golden film surface, finish the measurement of an ampicillin content.
(5) prepare once more from 1250.0,625.0,312.5,156.2,78.1,39.0,19.5,9.7,4.9,2.4,0.0 μ gl
-1The milk sample of different ampicillin content repeats the experimental procedure of (3)～(4), and each sample is measured;
(6) in (3), (4), the measurement data that obtains of (5) step utilizes least square method (or higher order polynomial fits) to handle, and obtains corresponding variations in refractive index curve, sets up forecast model;
(7) milk sample of the unknown ampicillin content of measurement obtains corresponding variations in refractive index curve, according to the forecast model that step (6) is set up, the content of ampicillin in the qualitative analysis milk.
In technique scheme, when carrying out the surface plasma body resonant vibration refractometry, all adopt the hyperchannel difference measurement, promptly in a passage, feed deionized water as measuring basis, feed sample to be measured in another passage simultaneously, two groups of data that record are carried out difference processing, thereby eliminate or weaken the influence to measuring accuracy such as environmental factor.
Be example with the content that detects microbiotic-ampicillin in the milk below, set forth the detailed detection step of indirect measurement method of surface plasma body resonant vibration refractive index:
(1) utilize BSA absorption and amine to connect the golden film surface that antibody is tied to surface plasma resonance sensor,
As shown in Figure 2, at first, SPR gold film surface is handled, step is as follows:
Clean golden film surface 9 with alcohol swab, the Sensor section 6 around avoiding running into;
The BSA (bovine serum albumin) 10 that configuration 5mg/ml dilutes with phosphate buffer, PH7.4;
The BSA of 0.5ml is added EDC (1-(3-dimethylamino-propyl)-3-ethyl carbodiimide)/NHS (N-hydroxy-succinamide), and in the solution of (4: 1), room temperature was placed 5 minutes for 20 ℃;
10 μ l are had active BSA solution put into the golden film surface of cleaning 9, set level golden film surface 9, make the full surface of BSA exhibition uniformly, kept 15 minutes under the room temperature;
Clean golden film surface 9 with 20mM hydrochloric acid, then use flushing with clean water again, the surface is dried up with air or nitrogen;
The fourth disuccinic acid acid anhydride that adds 0.2g in 0.5DMSO (dimethyl sulfoxide) makes its dissolving, and the biphosphate that adds 50mM is again received salt solusion makes volume reach 10ml, adds several 5M NaOH at last again its pH value is reached about 6.5.
Fourth disuccinic acid acid anhydride is put on the surface 10 that scribbles BSA BSA carry out carboxylic acidization, room temperature kept 12 hours for 20 ℃.Forbid that in this process golden film surface 9 runs into any solid surface, thereby make 9 activation of golden film surface, form carboxylated antibody protein molecular layer 11, become the connection tie of golden film and antibody protein molecular layer 12.
Amine connects the step of (Amine-coupling) binding antibody:
Feed phosphate buffer (PBS) to the albumin layer that has carboxyl, form benchmark;
Utilize EDC, NHS (volume ratio 1: 1) that the carboxyl albumin layer on golden film surface is carried out activation processing (2-7min), form the activated protein layer that has the NHS ester;
Feed PBS flushing SPR surface;
Feed antibody (20-50 μ l/ml), thereby it is attached on the golden film uniformly, form antibody molecule albumin layer 12;
After feeding PBS, feed monoethanolamine (1M, PH8.5) again and handle albumin layer 7min, cover non-specific antibody;
Feed the PBS flushing, form surface measurements;
(2) initialization system in air makes the spr sensor optimization;
(3) in surface plasma resonance sensor gold film surface, feed deionized water by injection device, obtain the stable measuring basis of refractive index (1.333);
(4) preparation ampicillin content is 2500.0 μ gl
-1Milk sample, inject surface plasma resonance sensor, image data, result obtains the variations in refractive index data;
(5) by the NaOH solution of injection device, destroy the combination of antigen-antibody, slough the ampicillin of antibody layer absorption, make its surface regeneration, finish one-shot measurement to surface plasma resonance sensor feeding 30mM;
(6) prepare once more from 1250.0,625.0,312.5,156.2,78.1,39.0,19.5,9.7,4.9,2.4,0.0 μ gl
-1The milk sample of different ampicillin content repeats the experimental procedure of (4)～(5), and each sample is measured;
(7) in (4), (5), the measurement data that obtains of (6) step utilizes least square method (or higher order polynomial fits) to handle, and obtains corresponding variations in refractive index curve, sets up forecast model;
(8) milk sample of the unknown ampicillin content of measurement obtains corresponding variations in refractive index curve, according to the forecast model that step (6) is set up, analyzes the content of ampicillin in the milk.
(9) when antibody protein layer approach exhaustion, remove albumin layer, clean golden film surface, antagonist albumen is bound again.
In technique scheme, when carrying out the surface plasma body resonant vibration refractometry, all adopt the hyperchannel difference measurement, promptly in a passage, feed deionized water as measuring basis, feed sample to be measured in another passage simultaneously, two groups of data that record are carried out difference processing, thereby eliminate or weaken the influence to measuring accuracy such as environmental factor.
The present invention is accurate and convenient for what narrate, all be that example is described in detail in an embodiment with the ampicillin, but the present invention is equally applicable to other penicillins, as benzyl penicillin by after selecting the antibody that adapts with microbiotic to be measured for use; Tetracyclines is as tetracycline, aureomycin, terramycin; Aminoglycoside is as gentamicin, streptomysin, kanamycins; The accurate detection and the qualitative analysis of aflatoxins equal size, so foregoing is all within protection domain of the present invention.
As shown in Figure 1, of the present invention based on detection of antibiotics system in the surface plasma body resonant vibration dairy products, include: be used for detecting golden film surface liquid change of refractive, gather spr sensor 6 parts of corresponding data; Be used for analyzing the data that spr sensor part 6 is gathered, the data analysis of gathering is handled, draw the data processing section 1 of the size of corresponding curve and antibiotic concentration; Be used for showing intuitively the display device part 3 of measurement result and corresponding measuring condition,, measure the demonstration of temperature, flow rate of liquid as the demonstration of antibiotic concentration; With constant rate of speed reagent is injected the injection device part 4 of spr sensor part 6 on request by reversal valve 5; Undertaken by the order of setting in order to control above-mentioned various piece, realize the control section 2 of the automatic measurement of sample; Collect the waste liquid pool 7 of waste liquid; Wherein, control section 2 links to each other with data processing section 1, display device part 3, injection device part 4, reversal valve 5, spr sensor part 6 respectively, and injection device part 4, reversal valve 5, spr sensor part 6, waste liquid pool 7 link to each other successively.
In said system, also be provided with by the golden film surface of reversal valve 5 in spr sensor part 6 and inject a kind of of strong acid and highly basic, golden film surface is cleaned, golden film surface capable of repeatedly is used realize the repeatedly cleaning device of precision measurement sample, the liquid feeding end of described cleaning device links to each other with control section 2, by the flow of control section 2 controlling liquid, outlet end links to each other with reversal valve 5.
In said system: injection device, realize that fluids such as milk sample, antibody protein and reagent inject surface plasma resonance sensor by ad hoc fashion as required; Reversal valve is realized the feeding control of each reagent, makes all ingredients feed the sample cell of surface plasma resonance sensor on request, in order; Utilize surface plasma resonance sensor, change of refractive such as the reagent that feeds sample cell, antibody, milk sample are carried out real-time, high-precision collecting test; Data handling system realizes the variations in refractive index according to specimen, sets up forecast model, calculates antibiotic content in the milk sample to be tested, and by display device measurement result is shown; Control device control each several part is realized the robotization control to injection device, reversal valve, display device and data handling system.
Described spr sensor part 6 is formed with the fluid passage 8 more than 1 or 1, measures simultaneously in order to realize several samples.Reversal valve 5 adopts the hyperchannel reversal valve.Use hyperchannel reversal valve 5 to realize different passages sample introduction simultaneously, the different sample rooms of same passage switch.During measurement, by each funtion part coordination in control device 2 control system, a passage feeds deionized water therein, and as the difference compensation channels of measuring, passage feeds sample reagent in addition, simultaneously a plurality of passages is measured, and obtains refractive index curve.
Because it is responsive that 6 pairs of air of spr sensor part and extraneous parasitic light all compare, and all seals in order to improve the whole device each several part of precision, removed in the pipeline before the operation and the air in the sample, and the measurement of total system is all carried out in the lucifuge environment.
Although combination of the present invention and method are described by detailed implementation process, but those skilled in the art obviously can be spliced the described method and apparatus of the application in not breaking away from content of the present invention, spirit and scope or change, or increase and decrease some parts, more particularly, the replacement that all are similar and change apparent to those skilled in the artly, they are regarded as being included among spirit of the present invention, scope and the content.
Claims (12)
1. one kind based on detection of antibiotics method in the surface plasma body resonant vibration dairy products, it is characterized in that, be to utilize the SPR optical means, measure the refractive index of the antibiotic dairy produce of known different content, and it is the size of the refractive index that measures is corresponding with antibiotics in milk products content, the refractive index of the refractive index of the dairy produce sample of the unknown antibiotic concentration that will measure and the antibiotic dairy produce of known different content compares again, realizes the detection of antibiotic content in the dairy produce.
2. according to claim 1 based on detection of antibiotics method in the surface plasma body resonant vibration dairy products, it is characterized in that, be a kind of method that adopts in the indirect measurement method of the direct measuring method of surface plasma body resonant vibration refractive index and surface plasma body resonant vibration refractive index, realize detection antibiotic content in the dairy produce.
3. according to claim 2 based on detection of antibiotics method in the surface plasma body resonant vibration dairy products, it is characterized in that, the direct measuring method of described surface plasma body resonant vibration refractive index, be directly to feed testing sample on the golden film surface of SPR, make sample directly contact golden film surface and measure, thereby realize antibiotics in milk products qualitative detection and analysis.
4. according to claim 3ly it is characterized in that based on detection of antibiotics method in the surface plasma body resonant vibration dairy products direct measuring method of described surface plasma body resonant vibration refractive index includes following steps:
(1) feeds deionized water, obtain the stable measuring basis of refractive index;
(2) the dairy produce sample of the known different antibiotic contents of preparation by surface plasmon resonance measurement amount refractive index size, obtains the pairing refractive index value of different antibiotic concentrations;
(3) set up the typical curve of antibiotic content in the dairy produce sample, and application of mathematical method sets up the theoretical model of antibiotic content, form forecast model;
(4) refractive index of the dairy produce of the unknown antibiotic content of measurement, measured result is brought into the forecast model of in the 3rd step, setting up, calculate antibiotic content wherein, and also at the otherness between the different dairy produces, and when test environment temperature and the otherness of forecast model temperature revise, thereby predicted the outcome.
5. according to claim 2 based on detection of antibiotics method in the surface plasma body resonant vibration dairy products, it is characterized in that, the indirect measurement method of described surface plasma body resonant vibration refractive index, after being antibody by microbiotic selective adsorption to be measured in golden film surface is bound dairy produce, improve specificity, measure the refractive index of dairy produce again and obtain the microbiotic of dairy produce, realize that the accurate quantification of antibiotic content in the dairy produce detects.
6. according to claim 5ly it is characterized in that based on detection of antibiotics method in the surface plasma body resonant vibration dairy products indirect measurement method of described surface plasma body resonant vibration refractive index includes following steps:
(1) utilize BSA absorption, amine to connect the golden film surface that antibody is attached to SPR;
(2) feed deionized water, obtain the stable measuring basis of refractive index;
(3) the dairy produce sample of the known different antibiotic contents of preparation by surface plasmon resonance measurement amount refractive index size, obtains the pairing refractive index value of different antibiotic concentrations;
(4) set up the typical curve of antibiotic content in the dairy produce sample, and application of mathematical method sets up the theoretical model of antibiotic content, form forecast model;
(5) refractive index of the dairy produce of the unknown antibiotic content of measurement, measured result is brought into the forecast model of in the 4th step, setting up, calculate antibiotic content wherein, and also at the otherness between the different dairy produces, and when test environment temperature and the otherness of forecast model temperature revise, thereby predicted the outcome.
7. according to claim 6ly it is characterized in that the described BSA of utilization absorption, amine connects the golden film surface that antibody is attached to SPR, is to be finished by following steps based on detection of antibiotics method in the surface plasma body resonant vibration dairy products:
(1) utilizes the BSA physical adsorption characteristic, evenly be applied to golden film surface, add 4: 1 EDC, NHS, and utilize fourth disuccinic acid acid anhydride that BSA carry out carboxylic acidization, form the carboxyl albumin layer, become the connection tie of golden film and antibody protein;
(2) utilize EDC, NHS that the carboxyl albumin layer on golden film surface is handled, form NHS ester activated protein layer, make antibody capable and BSA carry out amine and connect, thereby be attached to uniformly on the golden film.
8. according to claim 4 or 6 described based on detection of antibiotics method in the surface plasma body resonant vibration dairy products, it is characterized in that, when the variations in refractive index of the dairy produce sample of measuring unknown antibiotic concentration, all adopt the hyperchannel difference measurement, promptly in a passage, feed deionized water as measuring basis, feed sample to be measured in another passage simultaneously, two groups of data that record are carried out difference processing, thereby the result is revised.
9. according to claim 4 or 6 described based on detection of antibiotics method in the surface plasma body resonant vibration dairy products, it is characterized in that, all ingredients, sample when experimentizing kept 15-30 minute in 20-25 ℃ water, to guarantee that identical temperature is arranged, reagent, sample feed among the SPR with speed 5～10 μ l/min speed during measurement, continue 5-10 minute, obtain best test curve.
10. the microbiotic detection system based on the dairy produce of surface plasma body resonant vibration is characterized in that, includes: be used for detecting golden film surface liquid change of refractive, gather the spr sensor part (6) of corresponding data; Be used for analyzing the data that spr sensor part (6) is gathered, the data analysis of gathering is handled, draw the data processing section (1) of the size of corresponding curve and antibiotic concentration; Be used for showing intuitively the display device part (3) of measurement result and corresponding measuring condition; With constant rate of speed reagent is injected the injection device part (4) of spr sensor part (6) on request by reversal valve (5); Undertaken by the order of setting in order to control above-mentioned various piece, realize the control section (2) of the automatic measurement of sample; Collect the waste liquid pool (7) of waste liquid; Wherein, control section (2) links to each other with data processing section (1), display device part (3), injection device part (4), reversal valve (5), spr sensor part (6) respectively, and injection device part (4), reversal valve (5), spr sensor part (6), waste liquid pool (7) link to each other successively.
11. the microbiotic detection system of the dairy produce based on surface plasma body resonant vibration according to claim 10, it is characterized in that, also be provided with by the golden film surface of reversal valve (5) in spr sensor part (6) and inject a kind of of strong acid and highly basic, golden film surface is cleaned, golden film surface capable of repeatedly is used realize the repeatedly cleaning device of precision measurement sample, the liquid feeding end of described cleaning device links to each other with control section (2), and outlet end links to each other with reversal valve (5).
12. the microbiotic detection system of the dairy produce based on surface plasma body resonant vibration according to claim 10, it is characterized in that, described spr sensor part (6) is formed with the fluid passage (8) more than 1 or 1, measures simultaneously in order to realize several samples.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101706440B (en) * | 2009-12-02 | 2011-12-07 | 昆明理工大学 | Detection method of belt-lactam antibiotics residues in milk |
| CN101692080B (en) * | 2009-10-12 | 2013-01-09 | 天津大学 | Method for detecting multiple antibiotic residues in dairy products |
| CN103097875A (en) * | 2010-05-27 | 2013-05-08 | 艾皮森泰克公司 | Improved method of sensor measurement |
| CN108982421A (en) * | 2018-06-15 | 2018-12-11 | 集美大学 | The analyzing detecting method of neomycinsulphate |
| CN109507151A (en) * | 2018-12-14 | 2019-03-22 | 湖南农业大学 | Tetracycline detection method based on Applications of surface plasmon resonance |
| CN109991419A (en) * | 2019-03-18 | 2019-07-09 | 浙江大学 | Nicotinic insecticide thiacloprid method for detecting residue based on SPR technique |
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2006
- 2006-12-28 CN CN 200610130646 patent/CN1987427A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101692080B (en) * | 2009-10-12 | 2013-01-09 | 天津大学 | Method for detecting multiple antibiotic residues in dairy products |
| CN101706440B (en) * | 2009-12-02 | 2011-12-07 | 昆明理工大学 | Detection method of belt-lactam antibiotics residues in milk |
| CN103097875A (en) * | 2010-05-27 | 2013-05-08 | 艾皮森泰克公司 | Improved method of sensor measurement |
| CN103097875B (en) * | 2010-05-27 | 2015-11-25 | 艾皮森泰克公司 | The sensor measurement improved |
| CN108982421A (en) * | 2018-06-15 | 2018-12-11 | 集美大学 | The analyzing detecting method of neomycinsulphate |
| CN108982421B (en) * | 2018-06-15 | 2021-12-17 | 集美大学 | Analysis and detection method of neomycin sulfate |
| CN109507151A (en) * | 2018-12-14 | 2019-03-22 | 湖南农业大学 | Tetracycline detection method based on Applications of surface plasmon resonance |
| CN109991419A (en) * | 2019-03-18 | 2019-07-09 | 浙江大学 | Nicotinic insecticide thiacloprid method for detecting residue based on SPR technique |
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